Glycoproteins are found in eukaryotes from microorganisms such as yeast to human, and are reported to be found in several bacteria in recent years. The functions of their sugar chains relate to stability and protease resistance of protein, and are necessary for folding for the formation of a higher-order structure. In addition, glycoproteins are known to control the interaction between proteins, and bind to lectin on the cell surface to cause signal transduction. The analysis of these glycoproteins requires cleavage of the sugar chains and determination of their structure. Peptide: N-glycanase and endo-β-N-acetylglucosaminidases are known as the enzymes which cleaves the N-linked sugar chain attached to asparagine residues. The latter endo-β-N-acetylglucosaminidases are enzymes which cleave the bond between chitobiose molecules at the reducing ends of N-type sugar chains, and known examples include Arthrobacter-derived Endo-A (Non Patent Literature 1, Patent Literature 4), Streptococcus pneumoniae-derived Endo-D (Non Patent Literature 2), Flavobacterium-derived Endo-F (Non Patent Literature 3), Streptomyces plicatus-derived Endo-H (Non Patent Literature 4), Mycosphaerella-derived endo-β-N-acetylglucosaminidase (Patent Literature 3), rice-derived Endo-Os (Non Patent Literature 5), Mucor hiemalis-derived Endo-M (Patent Literatures 1, 2, and 5, Non Patent Literatures 6 and 7) are known. Many of them have digestion activity between chitobiose molecules, and transglycosidase activity for transferring sugar chains. More specifically, they efficiently catalyze the reaction including the action on the N-type sugar chain of a glycoprotein to cut out the sugar chain, and transfer of the sugar chain to a carbohydrate or complex carbohydrate as the acceptor. Accordingly, endo-β-N-acetylglucosaminidases are enzymes useful not only for the analysis of the sugar chain structure of glycoproteins, and also for the modification of glycoproteins and glycolipids, preparation of neoglycoproteins, and homogenization of the sugar chain of glycoproteins.
The asparagine-linked sugar chains of glycoproteins showing major biological activity are classified into high-mannose type (mannan type sugar chain), hybrid type, and complex type sugar chains, according to their structures. However, among the endo-β-N-acetylglucosaminidases, Endo-M, Endo-F2, Endo-F3, Endo-S, and Endo-CE are reported to have activity for cleaving the complex type sugar chain.
The properties of Endo-M are studied in detail, and its substrate specificity is 4.4% for the biantennary complex type sugar chain (agalacto biantennary PA-sugar) when the activity for the high-mannose type Man8GlcNAc2 is set at 100% (Non Patent Literature 6). In addition, there is a description that Endo-M can cleave the triantennary and asialo tetraantennary N-type sugar chains (Non Patent Literature 7), but the activity for asialo triantennary and asialo tetraantennary was not detected in the enzyme activity measurement using a PA sugar chain (Non Patent Literature 6). Endo-M also cannot cleave the biantennary PA-sugar chain to which core fucose is attached.
Endo-F2 is an enzyme derived from Elizabethkingia miricola, and hydrolyzes high-mannose- and biantennary complex type sugar chains, but has no activity for hydrolyzing a hybrid type sugar chain (Non Patent Literature 8). Endo-F3 is also an enzyme derived from Elizabethkingia miricola, and hydrolyzes a biantennary or triantennary complex type sugar chain, but has no activity for hydrolyzing high-mannose and hybrid type sugar chains (Non Patent Literature 8). Endo-S is an enzyme derived from Streptococcus pyogenes, and hydrolyzes a biantennary complex type sugar chain, but has no activity for hydrolyzing high-mannose type and hybrid type sugar chains (Non Patent Literature 9). Endo-CE is an enzyme derived from Caenorhabditis elegans, and hydrolyzes high-mannose and biantennary complex type sugar chains. However, it is unknown whether it cleaves a hybrid type sugar chain (Non Patent Literature 10).
Regarding the modification of a complex type sugar chain, based on prior art findings, the substrate specificity of transglycosidase activity of endo-β-N-acetylglucosaminidases is the same as their digestion activity, so only these enzymes can transfer complex type sugar chains to acceptors.
The provision of an endo-β-N-acetylglucosaminidase having different substrate specificity from Endo-M is desired for the analysis of the sugar chain structure of a glycoprotein and the synthesis of glycoproteins having various sugar chains including complex type carbohydrate sugar chains, and the enzyme having high specific activity for a complex type sugar chain is also desired.